Neonatal i.c.v. Injection of ASO and Mouse Tissue Collection

For neonatal i.c.v. injections, pups (P1–P2) were treated with a single 40-μg dose of 2′-O-methoxyethyl sugars (2′MOE) and a phosphorothioate (PS) backbone-modified NT ASO (5′-CCTCTTACCTCAGTTACA-3′) or ASO 41-1 (5′-CCTGATCACCTACCTGGT-3′) (Integrated DNA Technologies) via i.c.v. injection according to a published procedure.22 In brief, ASOs were diluted in sterile 0.9% saline with 0.01% Fast Green FCF, and 2.5 μL was injected into the left ventricle using a 33G needle (point style 4; angle, 30) affixed to a glass Hamilton syringe approximately 2.5 mm anterior to the lambda suture and 1 mm lateral to the sagittal suture to a depth of 2 mm. For the P21 time point, pups were treated with 25 μg of PMO NT ASO or 20 μg of 2′MOE ASO 41-1.

Transgenic hemizygote LRRK2 WT and LRRK2 G2019S BAC overexpressing animals were perfused with cold phosphate-buffered saline (PBS) either 3 weeks or 2 months after ASO i.c.v. injection. The brain was isolated and the left hemisphere was postfixed in 4% paraformaldehyde overnight and moved to 30% sucrose. The right hemisphere was immediately dissected and hippocampus, striatum, midbrain, and cortex tissue were all isolated, snap-frozen, and stored for analysis of exon skipping and protein expression.